Role of CYP2E1 in TNFa-Ethanol -induced Liver Injury

描述(申请人提供)：酒精导致细胞损伤的机制(S)尚不清楚。其中一个重要的机制是脂质过氧化和氧化应激在酒精毒性中的作用，这是相当多研究的焦点。许多途径被认为在乙醇诱导“氧化应激”的过程中起着关键作用，包括线粒体功能障碍、MAP激活酶的激活、乙醇诱导的细胞因子的增加，尤其是TNF1和细胞色素P450_2的诱导。这些途径并不是相互排斥的，但它们之间的联系和相互作用，特别是在体内条件下，仍有待评估和澄清。我们假设，体内诱导的氧化应激增加会使肝细胞对TNF1诱导的肝毒性反应敏感。我们认为，过氧亚硝酸盐等氧化剂、JNK和/或P38MAPK等MAP激酶的激活、NF-kB的失活和线粒体功能障碍是CYP2E1-TNF1增强肝毒性的下游介质。我们还假设，在体内酒精处理增强TNF1肝毒性的机制中，CYP2E1的诱导起着核心作用。由于CYP2E1和TNF1被认为是酒精性肝损伤发生的关键危险因素，因此对它们在体内可能的相互作用和增强作用进行评估是很重要的。目的1通过诱导细胞色素P450 2 1的表达，探讨其对肿瘤坏死因子1诱导的肝损伤的保护作用。TNF1或生理盐水将被注射到基础水平为CYP2E1的生理盐水处理的小鼠，或高水平的吡唑治疗的小鼠，以及没有CYP2E1的CYP2E1基因敲除小鼠。给小鼠注射不同浓度的TNF1，在不同时间处死小鼠。我们将检测以下几个方面：肝毒性、氧化/亚硝化应激、核因子-kB激活、MAP激酶激活、线粒体功能障碍。目的2评价细胞色素P450 2在乙醇增强肿瘤坏死因子1诱导的肝损伤中的作用，并确定其相互作用的下游靶点。急性酒精中毒模型的评估是先大量饮酒，然后再用TNF1或生理盐水激发。对于慢性乙醇喂养，小鼠将被喂以对照或乙醇Lieber-DeCarli饮食2、4或6周，然后用TNF1(或生理盐水)激发。将进行与上述类似的检测。为了证明在吡唑和乙醇模型中，细胞色素P450_2E_1对增强的损伤负责，将使用细胞色素P450_2E_1基因敲除小鼠和人源化的基因敲除小鼠。公共卫生相关性：我们相信这些实验将有助于阐明两个被认为对酒精性肝损伤起重要作用的独立风险因子--CYP2E1和TNF1是如何相互作用促进这种肝损伤的，将为CYP2E1在急性和慢性酒精/TNF1增强性肝损伤中的作用提供明确的证据，并有助于确定CYP2E1-乙醇-TNF1相互作用的下游介质、信号通路和靶点。后者可能具有潜在的治疗价值。PHS 398/2590(09/04版，2006年4月4日重新发布)页面续格式页面

DESCRIPTION (provided by applicant): The mechanism(s) by which alcohol causes cell injury are still not clear. A major mechanism that is a focus of considerable research is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a key role on how ethanol induces "oxidative stress", including mitochondrial dysfunction, activation of MAP kinases, ethanol-induced increases in cytokine formation, especially TNF1 and induction of CYP2E1. These pathways are not exclusive of each other, however, associations and interactions between them, especially under in vivo conditions, remain to be evaluated and clarified. We hypothesize that increased oxidative stress from CYP2E1 induction in vivo sensitizes hepatocytes to TNF1-induced hepatotoxicity. We propose that oxidants such as peroxynitrite, activation of MAP kinases such as JNK and/or P38 MAPK, inactivation of NF-kB and mitochondrial dysfunction are downstream mediators of the CYP2E1- TNF1 potentiated hepatotoxicity. We also hypothesize that induction of CYP2E1 plays a central role in mechanisms by which in vivo treatment with alcohol potentiates TNF1 hepatotoxicity. Since CYP2E1 and TNF1 are considered key risk factors in the development of alcoholic liver injury, possible interactions and potentiation of their actions in vivo is important to evaluate. AIM 1 will evaluate the potentiation of TNF1-induced liver injury by induction of CYP2E1. TNF1 or saline will be administrated to either saline-treated mice with basal levels of CYP2E1, or pyrazole-treated mice with high levels of CYP2E1, and to CYP2E1-knockout mice with no CYP2E1. Various concentrations of TNF1 will be given and mice killed at various times after TNF1 challenge. We will assay for the following: Hepatotoxicity, oxidative/nitrosative stress, activation of NF-kB, activation of MAP kinases, mitochondrial dysfunction. AIM 2 will evaluate the role of CYP2E1 in the potentiation of TNF1-induced liver injury by ethanol and to identify downstream targets for the CYP2E1-ethanol- TNF1 interactions. A binge model of alcohol intake followed by TNF1 or saline challenge will be the acute ethanol model evaluated. For chronic ethanol feeding, mice will be fed a control or an ethanol Lieber-DeCarli diet for 2, 4 or 6 weeks, followed by challenge with TNF1 (or saline). Assays similar to the above will be carried out. To prove that CYP2E1 is responsible for the potentiated injury in the pyrazole and ethanol models, CYP2E1 knockout mice and humanized CYP2E1 knockin mice will be used. PUBLIC HEALTH RELEVANCE: We believe that these experiments will help to clarify how two independent risk factors believed to be important for alcohol-induced liver injury, CYP2E1 and TNF1 interact to promote this liver injury, will provide clear evidence for a role for CYP2E1 in acute and chronic alcohol/ TNF1 potentiated liver injury, and help to identify downstream mediators, signaling pathways and targets of the CYP2E1-ethanol- TNF1 interactions. The latter may have potential therapeutic value. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page